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September 22, 2019  |  

Recurrent structural variation, clustered sites of selection, and disease risk for the complement factor H (CFH) gene family.

Structural variation and single-nucleotide variation of the complement factor H (CFH) gene family underlie several complex genetic diseases, including age-related macular degeneration (AMD) and atypical hemolytic uremic syndrome (AHUS). To understand its diversity and evolution, we performed high-quality sequencing of this ~360-kbp locus in six primate lineages, including multiple human haplotypes. Comparative sequence analyses reveal two distinct periods of gene duplication leading to the emergence of four CFH-related (CFHR) gene paralogs (CFHR2 and CFHR4 ~25-35 Mya and CFHR1 and CFHR3 ~7-13 Mya). Remarkably, all evolutionary breakpoints share a common ~4.8-kbp segment corresponding to an ancestral CFHR gene promoter that has expanded independently throughout primate evolution. This segment is recurrently reused and juxtaposed with a donor duplication containing exons 8 and 9 from ancestral CFH, creating four CFHR fusion genes that include lineage-specific members of the gene family. Combined analysis of >5,000 AMD cases and controls identifies a significant burden of a rare missense mutation that clusters at the N terminus of CFH [P = 5.81 × 10-8, odds ratio (OR) = 9.8 (3.67-Infinity)]. A bipolar clustering pattern of rare nonsynonymous mutations in patients with AMD (P < 10-3) and AHUS (P = 0.0079) maps to functional domains that show evidence of positive selection during primate evolution. Our structural variation analysis in >2,400 individuals reveals five recurrent rearrangement breakpoints that show variable frequency among AMD cases and controls. These data suggest a dynamic and recurrent pattern of mutation critical to the emergence of new CFHR genes but also in the predisposition to complex human genetic disease phenotypes.


September 22, 2019  |  

Long-term microbiota and virome in a Zürich patient after fecal transplantation against Clostridium difficile infection.

Fecal microbiota transplantation (FMT) is an emerging therapeutic option for Clostridium difficile infections that are refractory to conventional treatment. FMT introduces fecal microbes into the patient’s intestine that prevent the recurrence of C. difficile, leading to rapid expansion of bacteria characteristic of healthy microbiota. However, the long-term effects of FMT remain largely unknown. The C. difficile patient described in this paper revealed protracted microbiota adaptation processes from 6 to 42 months post-FMT. Ultimately, bacterial communities were donor similar, suggesting sustainable stool engraftment. Since little is known about the consequences of transmitted viruses during C. difficile infection, we also interrogated virome changes. Our approach allowed identification of about 10 phage types per sample that represented larger viral communities, and phages were found to be equally abundant in the cured patient and donor. The healthy microbiota appears to be characterized by low phage abundance. Although viruses were likely transferred, the patient established a virome distinct from the donor. Surprisingly, the patient had sequences of algal giant viruses (chloroviruses) that have not previously been reported for the human gut. Chloroviruses have not been associated with intestinal disease, but their presence in the oropharynx may influence cognitive abilities. The findings suggest that the virome is an important indicator of health or disease. A better understanding of the role of viruses in the gut ecosystem may uncover novel microbiota-modulating therapeutic strategies.© 2016 New York Academy of Sciences.


September 22, 2019  |  

Sixteen diverse laboratory mouse reference genomes define strain-specific haplotypes and novel functional loci.

We report full-length draft de novo genome assemblies for 16 widely used inbred mouse strains and find extensive strain-specific haplotype variation. We identify and characterize 2,567 regions on the current mouse reference genome exhibiting the greatest sequence diversity. These regions are enriched for genes involved in pathogen defence and immunity and exhibit enrichment of transposable elements and signatures of recent retrotransposition events. Combinations of alleles and genes unique to an individual strain are commonly observed at these loci, reflecting distinct strain phenotypes. We used these genomes to improve the mouse reference genome, resulting in the completion of 10 new gene structures. Also, 62 new coding loci were added to the reference genome annotation. These genomes identified a large, previously unannotated, gene (Efcab3-like) encoding 5,874 amino acids. Mutant Efcab3-like mice display anomalies in multiple brain regions, suggesting a possible role for this gene in the regulation of brain development.


September 22, 2019  |  

High-resolution comparative analysis of great ape genomes.

Genetic studies of human evolution require high-quality contiguous ape genome assemblies that are not guided by the human reference. We coupled long-read sequence assembly and full-length complementary DNA sequencing with a multiplatform scaffolding approach to produce ab initio chimpanzee and orangutan genome assemblies. By comparing these with two long-read de novo human genome assemblies and a gorilla genome assembly, we characterized lineage-specific and shared great ape genetic variation ranging from single- to mega-base pair-sized variants. We identified ~17,000 fixed human-specific structural variants identifying genic and putative regulatory changes that have emerged in humans since divergence from nonhuman apes. Interestingly, these variants are enriched near genes that are down-regulated in human compared to chimpanzee cerebral organoids, particularly in cells analogous to radial glial neural progenitors. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.


September 22, 2019  |  

Genome-wide characterization of human L1 antisense promoter-driven transcripts.

Long INterspersed Element-1 (LINE-1 or L1) is the only autonomously active, transposable element in the human genome. L1 sequences comprise approximately 17 % of the human genome, but only the evolutionarily recent, human-specific subfamily is retrotransposition competent. The L1 promoter has a bidirectional orientation containing a sense promoter that drives the transcription of two proteins required for retrotransposition and an antisense promoter. The L1 antisense promoter can drive transcription of chimeric transcripts: 5′ L1 antisense sequences spliced to the exons of neighboring genes.The impact of L1 antisense promoter activity on cellular transcriptomes is poorly understood. To investigate this, we analyzed GenBank ESTs for messenger RNAs that initiate in the L1 antisense promoter. We identified 988 putative L1 antisense chimeric transcripts, 911 of which have not been previously reported. These appear to be alternative genic transcripts, sense-oriented with respect to gene and initiating near, but typically downstream of, the gene transcriptional start site. In multiple cell lines, L1 antisense promoters display enrichment for YY1 transcription factor and histone modifications associated with active promoters. Global run-on sequencing data support the activity of the L1 antisense promoter. We independently detected 124 L1 antisense chimeric transcripts using long read Pacific Biosciences RNA-seq data. Furthermore, we validated four chimeric transcripts by quantitative RT-PCR and Sanger sequencing and demonstrated that they are readily detectable in many normal human tissues.We present a comprehensive characterization of human L1 antisense promoter-driven transcripts and provide substantial evidence that they are transcribed in a variety of human cell-types. Our findings reveal a new wide-reaching aspect of L1 biology by identifying antisense transcripts affecting as many as 4 % of all human genes.


September 22, 2019  |  

Next generation sequencing technology: Advances and applications.

Impressive progress has been made in the field of Next Generation Sequencing (NGS). Through advancements in the fields of molecular biology and technical engineering, parallelization of the sequencing reaction has profoundly increased the total number of produced sequence reads per run. Current sequencing platforms allow for a previously unprecedented view into complex mixtures of RNA and DNA samples. NGS is currently evolving into a molecular microscope finding its way into virtually every fields of biomedical research. In this chapter we review the technical background of the different commercially available NGS platforms with respect to template generation and the sequencing reaction and take a small step towards what the upcoming NGS technologies will bring. We close with an overview of different implementations of NGS into biomedical research. This article is part of a Special Issue entitled: From Genome to Function. Copyright © 2014 Elsevier B.V. All rights reserved.


September 22, 2019  |  

Comparative Annotation Toolkit (CAT)-simultaneous clade and personal genome annotation.

The recent introductions of low-cost, long-read, and read-cloud sequencing technologies coupled with intense efforts to develop efficient algorithms have made affordable, high-quality de novo sequence assembly a realistic proposition. The result is an explosion of new, ultracontiguous genome assemblies. To compare these genomes, we need robust methods for genome annotation. We describe the fully open source Comparative Annotation Toolkit (CAT), which provides a flexible way to simultaneously annotate entire clades and identify orthology relationships. We show that CAT can be used to improve annotations on the rat genome, annotate the great apes, annotate a diverse set of mammals, and annotate personal, diploid human genomes. We demonstrate the resulting discovery of novel genes, isoforms, and structural variants-even in genomes as well studied as rat and the great apes-and how these annotations improve cross-species RNA expression experiments.© 2018 Fiddes et al.; Published by Cold Spring Harbor Laboratory Press.


September 22, 2019  |  

A comparative transcriptional landscape of maize and sorghum obtained by single-molecule sequencing.

Maize and sorghum are both important crops with similar overall plant architectures, but they have key differences, especially in regard to their inflorescences. To better understand these two organisms at the molecular level, we compared expression profiles of both protein-coding and noncoding transcripts in 11 matched tissues using single-molecule, long-read, deep RNA sequencing. This comparative analysis revealed large numbers of novel isoforms in both species. Evolutionarily young genes were likely to be generated in reproductive tissues and usually had fewer isoforms than old genes. We also observed similarities and differences in alternative splicing patterns and activities, both among tissues and between species. The maize subgenomes exhibited no bias in isoform generation; however, genes in the B genome were more highly expressed in pollen tissue, whereas genes in the A genome were more highly expressed in endosperm. We also identified a number of splicing events conserved between maize and sorghum. In addition, we generated comprehensive and high-resolution maps of poly(A) sites, revealing similarities and differences in mRNA cleavage between the two species. Overall, our results reveal considerable splicing and expression diversity between sorghum and maize, well beyond what was reported in previous studies, likely reflecting the differences in architecture between these two species.© 2018 Wang et al.; Published by Cold Spring Harbor Laboratory Press.


September 22, 2019  |  

Young genes have distinct gene structure, epigenetic profiles, and transcriptional regulation.

Species-specific, new, or “orphan” genes account for 10%-30% of eukaryotic genomes. Although initially considered to have limited function, an increasing number of orphan genes have been shown to provide important phenotypic innovation. How new genes acquire regulatory sequences for proper temporal and spatial expression is unknown. Orphan gene regulation may rely in part on origination in open chromatin adjacent to preexisting promoters, although this has not yet been assessed by genome-wide analysis of chromatin states. Here, we combine taxon-rich nematode phylogenies with Iso-Seq, RNA-seq, ChIP-seq, and ATAC-seq to identify the gene structure and epigenetic signature of orphan genes in the satellite model nematode Pristionchus pacificus Consistent with previous findings, we find young genes are shorter, contain fewer exons, and are on average less strongly expressed than older genes. However, the subset of orphan genes that are expressed exhibit distinct chromatin states from similarly expressed conserved genes. Orphan gene transcription is determined by a lack of repressive histone modifications, confirming long-held hypotheses that open chromatin is important for new gene formation. Yet orphan gene start sites more closely resemble enhancers defined by H3K4me1, H3K27ac, and ATAC-seq peaks, in contrast to conserved genes that exhibit traditional promoters defined by H3K4me3 and H3K27ac. Although the majority of orphan genes are located on chromosome arms that contain high recombination rates and repressive histone marks, strongly expressed orphan genes are more randomly distributed. Our results support a model of new gene origination by rare integration into open chromatin near enhancers.© 2018 Werner et al.; Published by Cold Spring Harbor Laboratory Press.


September 22, 2019  |  

Transcriptional fates of human-specific segmental duplications in brain.

Despite the importance of duplicate genes for evolutionary adaptation, accurate gene annotation is often incomplete, incorrect, or lacking in regions of segmental duplication. We developed an approach combining long-read sequencing and hybridization capture to yield full-length transcript information and confidently distinguish between nearly identical genes/paralogs. We used biotinylated probes to enrich for full-length cDNA from duplicated regions, which were then amplified, size-fractionated, and sequenced using single-molecule, long-read sequencing technology, permitting us to distinguish between highly identical genes by virtue of multiple paralogous sequence variants. We examined 19 gene families as expressed in developing and adult human brain, selected for their high sequence identity (average >99%) and overlap with human-specific segmental duplications (SDs). We characterized the transcriptional differences between related paralogs to better understand the birth-death process of duplicate genes and particularly how the process leads to gene innovation. In 48% of the cases, we find that the expressed duplicates have changed substantially from their ancestral models due to novel sites of transcription initiation, splicing, and polyadenylation, as well as fusion transcripts that connect duplication-derived exons with neighboring genes. We detect unannotated open reading frames in genes currently annotated as pseudogenes, while relegating other duplicates to nonfunctional status. Our method significantly improves gene annotation, specifically defining full-length transcripts, isoforms, and open reading frames for new genes in highly identical SDs. The approach will be more broadly applicable to genes in structurally complex regions of other genomes where the duplication process creates novel genes important for adaptive traits.© 2018 Dougherty et al.; Published by Cold Spring Harbor Laboratory Press.


September 22, 2019  |  

Emergence, retention and selection: A trilogy of origination for functional de novo proteins from ancestral lncRNAs in primates.

While some human-specific protein-coding genes have been proposed to originate from ancestral lncRNAs, the transition process remains poorly understood. Here we identified 64 hominoid-specific de novo genes and report a mechanism for the origination of functional de novo proteins from ancestral lncRNAs with precise splicing structures and specific tissue expression profiles. Whole-genome sequencing of dozens of rhesus macaque animals revealed that these lncRNAs are generally not more selectively constrained than other lncRNA loci. The existence of these newly-originated de novo proteins is also not beyond anticipation under neutral expectation, as they generally have longer theoretical lifespan than their current age, due to their GC-rich sequence property enabling stable ORFs with lower chance of non-sense mutations. Interestingly, although the emergence and retention of these de novo genes are likely driven by neutral forces, population genetics study in 67 human individuals and 82 macaque animals revealed signatures of purifying selection on these genes specifically in human population, indicating a proportion of these newly-originated proteins are already functional in human. We thus propose a mechanism for creation of functional de novo proteins from ancestral lncRNAs during the primate evolution, which may contribute to human-specific genetic novelties by taking advantage of existed genomic contexts.


September 22, 2019  |  

RNAi-based treatment of chronically infected patients and chimpanzees reveals that integrated hepatitis B virus DNA is a source of HBsAg.

Chronic hepatitis B virus (HBV) infection is a major health concern worldwide, frequently leading to liver cirrhosis, liver failure, and hepatocellular carcinoma. Evidence suggests that high viral antigen load may play a role in chronicity. Production of viral proteins is thought to depend on transcription of viral covalently closed circular DNA (cccDNA). In a human clinical trial with an RNA interference (RNAi)-based therapeutic targeting HBV transcripts, ARC-520, HBV S antigen (HBsAg) was strongly reduced in treatment-naïve patients positive for HBV e antigen (HBeAg) but was reduced significantly less in patients who were HBeAg-negative or had received long-term therapy with nucleos(t)ide viral replication inhibitors (NUCs). HBeAg positivity is associated with greater disease risk that may be moderately reduced upon HBeAg loss. The molecular basis for this unexpected differential response was investigated in chimpanzees chronically infected with HBV. Several lines of evidence demonstrated that HBsAg was expressed not only from the episomal cccDNA minichromosome but also from transcripts arising from HBV DNA integrated into the host genome, which was the dominant source in HBeAg-negative chimpanzees. Many of the integrants detected in chimpanzees lacked target sites for the small interfering RNAs in ARC-520, explaining the reduced response in HBeAg-negative chimpanzees and, by extension, in HBeAg-negative patients. Our results uncover a heretofore underrecognized source of HBsAg that may represent a strategy adopted by HBV to maintain chronicity in the presence of host immunosurveillance. These results could alter trial design and endpoint expectations of new therapies for chronic HBV. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.


September 22, 2019  |  

Long-read sequencing of human cytomegalovirus transcriptome reveals RNA isoforms carrying distinct coding potentials.

The human cytomegalovirus (HCMV) is a ubiquitous, human pathogenic herpesvirus. The complete viral genome is transcriptionally active during infection; however, a large part of its transcriptome has yet to be annotated. In this work, we applied the amplified isoform sequencing technique from Pacific Biosciences to characterize the lytic transcriptome of HCMV strain Towne varS. We developed a pipeline for transcript annotation using long-read sequencing data. We identified 248 transcriptional start sites, 116 transcriptional termination sites and 80 splicing events. Using this information, we have annotated 291 previously undescribed or only partially annotated transcript isoforms, including eight novel antisense transcripts and their isoforms, as well as a novel transcript (RS2) in the short repeat region, partially antisense to RS1. Similarly to other organisms, we discovered a high transcriptional diversity in HCMV, with many transcripts only slightly differing from one another. Comparing our transcriptome profiling results to an earlier ribosome footprint analysis, we have concluded that the majority of the transcripts contain multiple translationally active ORFs, and also that most isoforms contain unique combinations of ORFs. Based on these results, we propose that one important function of this transcriptional diversity may be to provide a regulatory mechanism at the level of translation.


September 22, 2019  |  

De novo assembly of a Chinese soybean genome.

Soybean was domesticated in China and has become one of the most important oilseed crops. Due to bottlenecks in their introduction and dissemination, soybeans from different geographic areas exhibit extensive genetic diversity. Asia is the largest soybean market; therefore, a high-quality soybean reference genome from this area is critical for soybean research and breeding. Here, we report the de novo assembly and sequence analysis of a Chinese soybean genome for “Zhonghuang 13” by a combination of SMRT, Hi-C and optical mapping data. The assembled genome size is 1.025 Gb with a contig N50 of 3.46 Mb and a scaffold N50 of 51.87 Mb. Comparisons between this genome and the previously reported reference genome (cv. Williams 82) uncovered more than 250,000 structure variations. A total of 52,051 protein coding genes and 36,429 transposable elements were annotated for this genome, and a gene co-expression network including 39,967 genes was also established. This high quality Chinese soybean genome and its sequence analysis will provide valuable information for soybean improvement in the future.


September 22, 2019  |  

Isoform evolution in primates through independent combination of alternative RNA processing events.

Recent RNA-seq technology revealed thousands of splicing events that are under rapid evolution in primates, whereas the reliability of these events, as well as their combination on the isoform level, have not been adequately addressed due to its limited sequencing length. Here, we performed comparative transcriptome analyses in human and rhesus macaque cerebellum using single molecule long-read sequencing (Iso-seq) and matched RNA-seq. Besides 359 million RNA-seq reads, 4,165,527 Iso-seq reads were generated with a mean length of 14,875?bp, covering 11,466 human genes, and 10,159 macaque genes. With Iso-seq data, we substantially expanded the repertoire of alternative RNA processing events in primates, and found that intron retention and alternative polyadenylation are surprisingly more prevalent in primates than previously estimated. We then investigated the combinatorial mode of these alternative events at the whole-transcript level, and found that the combination of these events is largely independent along the transcript, leading to thousands of novel isoforms missed by current annotations. Notably, these novel isoforms are selectively constrained in general, and 1,119 isoforms have even higher expression than the previously annotated major isoforms in human, indicating that the complexity of the human transcriptome is still significantly underestimated. Comparative transcriptome analysis further revealed 502 genes encoding selectively constrained, lineage-specific isoforms in human but not in rhesus macaque, linking them to some lineage-specific functions. Overall, we propose that the independent combination of alternative RNA processing events has contributed to complex isoform evolution in primates, which provides a new foundation for the study of phenotypic difference among primates.© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.


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